Inhibitors and substrates of thrombin

ABSTRACT

Peptides which act as inhibitors or substrates of thrombin are derived from the formula: D-Phe-Pro-Arg or its analogues, wherein Phe is substituted by (a), wherein Ar 1  or Ar 2  are the same or different and are selected from the group consisting of phenyl, thienyl, pyridyl, naphthyl, thionaphtyl, indolyl and saturated groups corresponding to these, optionally substituted by up to three groups selected from C 1  -C 3  alkyl and C 1  -C 3  alkoxy, L 1  and L 2  are the same of different and are selected from the group consisting of CH 2 , C 2  --C 2 , O-CH 2 , S-CH 2 , Ar-L taken together optionally means H, diphenyl-methyl, fluorenyl or saturated groups corresponding to these, but one of the Ar-L cannot be H when the other [CH 2  ] n  -Q, (c) where Q=H, amino, amidino, imidazole, guanidino or isothioureido and n=1-5, preferably 3-5, or C 3  -C 9  alkyl and C 5  -C 10  aryl or alkylaryl optionally substituted by up to three groups selected from hydroxy and C 1  -C 4  alkoxy; Z=CN, COR 1 , (d) or (e), where R 1  =H, OH, CH 2  Cl, CH 2  --C 2  --CO--pip, CF 2  --CF 2  --CO--pip, (f), (g), CH 2  --C 2  --CO--Pro--NHEt, CF 2  --CF 2  --CO--Pro--NHEt or a chromophoric group e.g. pHA, MCA, R 2  and R 3  are the same or different and are selected from the group consisting of OH, OR 6  and NR 6  R 7 , or R 2  and R 3  taken together represent the residue of a diol; where R 6  and R 7 , which are the same or different, are C 1  -C 10  alkyl, phenyl or C 6  -C 10  arylalkyl, R 4  and R 5  are the same or different and are selected from R 2 , R 3 , Gly-pip, Ala-pip or Gly-Pro-NHEt.

This is a continuation of application Ser. No. 08/158,046 filed Nov. 24,1993, and now abandoned, which is a continuation of application Ser. No.07/866,178, filed as PCT/GB91/01946, Nov. 6, 1991, and now abandoned,and earlier international applications of record.

This invention relates to thrombin inhibitors and substrates.

Thrombin, the last enzyme in the coagulation system, cleaves solublefibrinogen to fibrin, which is then crosslinked and forms an insolublegel forming the matrix for a thrombus. When a vessel is damaged, theabove process is necessary to stop bleeding. Under normal circumstancesthere is no measurable amount of thrombin present in plasma. Increase ofthe thrombin concentration can result in formation of clots, which canlead to thromboembolic disease, one of the most common serious medicalproblems of our time.

Thrombin contributes to haemostatic control by means of severalbiological reactions. In addition to its primary function, theconversion of fibrinogen to fibrin, thrombin activates Factor XIII,which is responsible for the crosslinking of fibrin. Thrombin also actsby means of a positive feedback mechanism involving the activation ofFactors V and VIII, which both are necessary for its own formulationfrom prothrombin. Thrombin has another essential role: its binding toplatelets initiates platelet release and aggregation which isresponsible for primary haemostasis.

Fibrinolysis is the process which causes an enzymatic dissolution offibrinogen and fibrin clots. Plasma contains a protein, plasminogen,which under the influence of various activators is converted to plasma,a proteolytic enzyme, the activity of which resembles that of fibrin.Plasmin breaks down fibrin to fibrin degradation products.

Under normal conditions, the fibrinolysis system is in balance with thecoagulation system. Small thrombi formed in the blood stream can bedissolved enzymatically and the circulation through the vessels can berestored by the activation of the fibrinolytic system in the body. Ifthe fibrinolytic activity is too high, it may cause or prolong bleedingand if it is too low compared to the activity of the coagulation system,there is a risk of thrombosis.

The reactions of thrombin are further controlled by natural inhibitorsin plasma. The most important of these are antithrombin III and heparin.These two compounds have been isolated and are therapeutically andprophylactically used in conditions where there is an imbalance in thehaemostatic mechanisms with risk for prothrombin activation.

Mainly two types of therapeutic agents are used for the prevention ofthrombosis. The heparins act by accelerating the inhibition of thrombinby antithrombin III. Coumarin derivatives, the oral anticoagulants, e.g.Warfarin, prevent the generation of thrombin by blocking thepost-translational vitamin K-dependent α-carboxylation in the synthesisof prothrombin. Neither Heparin nor Warfarin are ideal. Heparin must begiven parenterally and as it functions as a cofactor to antithrombin IIIit has no effect without this inhibitor. The effect of Warfarin developsvery slowly and individual doses must be adjusted by frequent tests.None of these anticoagulants is specific for thrombin, they also inhibitother serine proteases and both of them may cause bleeding if the dosesare not correctly balanced.

Thus, direct acting, specific thrombin inhibitors, having oral activitywould be useful alternatives to the above anticoagulants. Much researchin this area has resulted in the synthesis of different kinds ofinhibitors of thrombin.

By imitating amino acid sequences of fibrinogen, the important naturalsubstrate of thrombin, several good short peptide substrates forthrombin have been synthesized. The very first developed sequence withaffinity for the active site of thrombin was Phe-Val-Arg [1] whichmimics the fibrinogensequence preceding the bond split by thrombin. Thissequence has later been improved to give D-Phe-Pro-Arg and D-Phe-Pip-Argwhich have been used in chromogenic substrates, e.g. D-Phe-Pro-Arg-pNAand D-Phe-Pip-Arg-pNA[1] and in inhibitors of thrombin, e.g. the peptidealdehyde D-Phe-Pro-Arg-H [2], the irreversible inhibitorD-Phe-Pro-Arg-CH₂ Cl [3], inhibitors with a ketomethylene bond e.g.D-Phe-Pro-Arg-K-Gly-piperidide [4] and in the recently synthesizedpeptide boronic acid inhibitors e.g. Z-D-Phe-Pro-boroArg [5] and thenitrile: Boc-D-Phe-Pro-ArgCN [6].

Thus, D-Phe-Pro-Arg has been considered the best sequence for about 15years, and it has been shown to have very good affinity for the activesite of thrombin, in substrates (Km around 10⁻⁶ M) as well as ininhibitors (Ki 10⁻⁷ M to 10⁻⁹ M).

We have now found that by exchanging Phe, in the D-Phe-Pro-Arg sequence,for some unnatural, aromatic amino acids, with a specified structure,and by using these new sequences to construct novel substrates andinhibitors we obtained significantly improved substrate and inhibitorproperties. The new substrates show better kinetic constants (Km andkcat) and the inhibitors better inhibition constant (Ki).

Reduction of blood pressure is a side effect observed in many of theprevious thrombin inhibitors containing Arg or Arg analogues like Gpaand Apa [7]. This side effect which in some compounds can bedisturbingly serious is believed to depend on the positively chargedguanidino or amidino group of the side chain of Arg or its analogues.Surprisingly, this side effect of inhibitors in the present applicationis markedly reduced even when the inhibitors have an Arg or Arganalogue.

We have also surprisingly found that by changing the side chain to anon-basic alkyl or alkylaryl group of a certain size, the affinity forthrombin is still very good although the affinity for other serineproteases is greatly reduced, i.e. these inhibitors/substrates are morespecific for thrombin than corresponding compounds containing Arg. Withthis non-basic side chain the blood pressure lowering side effect isgreatly reduced.

The present invention provides thrombin inhibitors and substratesderived from D-Phe-Pro-Arg or its analogues wherein Phe is substitutedby ##STR1## and Arg may be substituted by ##STR2##

Suitably, the inhibitors/substrates are of formula I, in which ##STR3##X=H, CH₃ or an N-protecting group, e.g. Ac, Bz, Cbz, Boc; Y=[CH₂ ]_(n)-Q, ##STR4## where Q=H, amino, amidino, imidazole, guanidino orisothioureido and n=1-5, preferably 3-5, or C₃ -C₉ alkyl and C₅ -C₁₀aryl or alkylaryl optionally substituted by up to three groups selectedfrom hydroxy and C₁ -C₄ alkoxy:

Z=CN, COR₁, ##STR5## where R₁ =H, OH, CH₂ Cl, CH₂ 13 C₂ --CO--pip, CF₂--C₂ --CO--pip, ##STR6## CH₂ --CH₂ --CO--Pro--NHEt, CF₂ --CF₂--CO--Pro--NHEt or a chromophoric group e.g. pNA, MCA,

R₂ and R₃ may be the same or different and are selected from the groupconsisting of OH, OR₆ and NR₆ R₇, which may be the same or different,are C₁ -C₁₀ alkyl, phenyl or C₆ -C₁₀ arylalkyl,

R₄ and R₅ may be the same or different and are selected from R₂, R₃,Gly-pip, Ala-pip or Gly-Pro-NHEt; ##STR7## where Ar₁ and Ar₂ may be thesame or different and are selected from the group consisting of phenyl,thienyl, pyridyl, naphthyl, thionaphthyl, indolyl and saturated groupscorresponding to these, optionally substituted by up to three groupsselected from C₁ -C₃ alkyl and C₁ -C₃ alkoxy,

Lhd 1 and L₂ may be the same or different and are selected from thegroup consisting of CH₂, CH₂ --CH₂, O--CH₂, S--CH₂,

Ar-L taken together may mean H, diphenyl-methyl, fluorenyl or saturatedgroups corresponding to these, but on of the Ar-L cannot be H when theother Ar-L means H or benzyl; ##STR8## or its C₁ -C₃ alkyl substitutedderivatives, where R₈ =CH₂, CH₂ --C₂, S--CH₂, S--C(CH₃)₂ or CH₂ --CH₂--CH₂.

Preferably the Phe substitute is Dpa, Nal or Dba and preferable Argsubstitute includes Irg, Gpa, Apa and non-basic amino acids such as Pgl,Mbg, Chg.

Examples of compounds which may be preferably used in the inventioninclude:

Ac-D-βNal-Pro-boroArg pinanediol ester

Z-D-Dpa-Pro-boroIrg pinanediol ester

Z-D-Dpa-Pro-boroPgl pinacol ester

Ac-D-βNal-Pro-boroMbg pinanediol ester

CH₃ -D-Dpa-Pro-Arg-H

Boc-D-Dpa-Pro-Gpa-H

CH₃ D-Dpa-Thi-Mbg-H

H-D-Dpa-Pro-Arg-k-Gly-pip

Z-D-Dpa-Pro-Arg-CH₂ Cl

Boc-D-Dpa-Pro-ArgCN

H-Dpa-Pro-Arg^(P) (OPh)₂

H-D-βNal-Pro-Pgl^(P) (OPh)-Gly-pip

H-D-Dpa-Pip-Arg-pNA

H-D-βNal-Pro-Chg-pNA

Further examples of compounds which may be preferably used in theinvention are those listed in Examples 10 to 22 below.

Inhibition data for some of the new compounds are shown in Tables 1-7.The advantages of replacing Phe by amino acids according to theinvention are clearly shown in the Ki values, which are generally 3 to10 times better for the new compounds, as well as in the prolongation ofthe thrombin time. The importance of the D-form of the N-terminal aminoacid is also evident from Table 1. The drastic reduction of the bloodpressure lowering side-effect with compounds according to the inventionis shown in Table 2.

                  TABLE 1                                                         ______________________________________                                                               Ki     TT     APTT                                     In-vitroassays         (μM)                                                                              (μM)                                                                              (μM)                                  ______________________________________                                             D-Phe--Pro--Gpa--k----Gly--Pip                                                                      1.3    4.9                                         4    D,L-Dpa--Pro--Arg----k--Gly--Pip                                                                    0.6         45.0                                   6    D-Dpa--Pro--Arg----k--Gly--Pip                                                                      0.2    0.65 8.0                                    7    L,Dpa--Pro--Arg----k--Gly--Pip                                                                      1.7    7.5  100.0                                  8    D-Fgl--Pro--Arg----k----Gly--Pip                                                                           50.0                                        9    D,L-α--Nal--Pro--Arg----k--Gly--Pip                                                           13.5                                               14   D,L-β--Nal--Pro--Arg----k--Gly--Pip                                                            11.6   90.0                                        54   D-B--Nal--Pro--Arg----k--Gly--Pip                                                                   2.87   5.3  45.0                                   ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                                               Blood                                                                         pressure                                                        TT      APTT  % of                                   In-vitroassay    Ki(μM)                                                                             (μM)*                                                                              (μM)                                                                             normal                                 ______________________________________                                            Boc-D-Phe--Pro--Arg--H                                                                         0.1     5            40**                                    Boc-D,L-Dpa--Pro--Arg--H                                                                       0.03    3           100**                                    Boc-D,L-Dpa--Pro--Gpa--H                                                                       0.03    3           nd                                   33  Z-D-Phe--Pro--Pgl--H                                                                           4.66    86.0        nd                                   48  Z-D,L-Dpa--Pro--Pgl--H                                                                         0.071               nd                                   53  Boc-D-Phe--Pro--His--H                                                                         0.726   3.1   34.0  100***                               ______________________________________                                         *The concentration needed to double the plasma thrombin time                  **4 mg/Kg given i.v. to anaesthetized cats                                    ***1 mg/Kg given i.v. bolus to anaesthetized New Zealand white rabbits, 2     per point                                                                     ND = Not determined.                                                     

                  TABLE 3                                                         ______________________________________                                                          Ki (uM)                                                     ______________________________________                                        Boc-D-Phe--Pro--ArgCN                                                                             0.8                                                       "-c-D,L-Dpa         0.05                                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        In-vitroassay         Ki(μM)                                                                              TT(μM)                                      ______________________________________                                        15     Boc-D,L-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                           59.0                                                16     D,L-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                               0.223                                               17     Z-D-Dpa--Pro--Pgl.sup.P (OPh).sub.2                                                              8.46                                                18     D-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                                 15.1                                                19     Z-D-Phs--Pro--Pgl.sup.P (OPh).sub.2                                                              >31.0                                               20     D-Phe--Pro--Pgl.sup.P (OPh).sub.2                                                                0.109                                               21     D-Phe--Pro--Pgl.sup.P (OH).sub.2                                                                 >98.0                                               23     D-Dpa--Pro--Pgl.sup.P (OPh).sub.                                                                 0.48                                                32     H-D-Dpa--Pro--Mpg.sup.P (OPh).sub.2                                                              0.0048   9.1                                        40     H-D-Phe--Pro--Mpg.sup.P (OPh).sub.2                                                              0.0188                                              ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                                                             APTT                                     In-vitroassays      Ki(pM)*  TT(μM)                                                                             (μM)                                  ______________________________________                                        12  Z-D-Dpa--Pro--BoroIrg--OPin                                                                       368.0    0.84  1.19                                   26  Z-D-β--Nal--Pro--BoroIrg--OPin                                                               325.0    0.15  0.216                                  36  Z-D-Fgl--Pro--BoroIrg--OPin                                                                       1750.0   0.643 1.23                                   37  Ac-D-Dpa--Pro--BoroIrg--OPin                                                                      2320.0         0.156                                  38  Z-L-Dpa--Pro--BoroIrg--OPin                                                                       4180.0   0.26  0.239                                  46  Z-D-Cha--Pro--BoroIrg--OPin                                                                       1490.0   0.113 0.123                                  ______________________________________                                         *Inhibitor PreIncubated for 30 min with Thrombin.                        

                  TABLE 6                                                         ______________________________________                                                                             APTT                                     In-vitroassays       Ki(nM)  TT(μM)                                                                             (μM)                                  ______________________________________                                        22   Z-D-Phe--Pro--BoroMbg--OPin                                                                       7.0     0.56  4.11                                   40   Z-D-Phe--Pro--BoroPhe--OPinac                                                                     12.8    0.329 2.52                                   10   Z-D-Phe--Pro--BoroAcet--OPinac                                                                    25.0    3.4   18.2                                   11   Z-D-Phe--Pro--BoroPgl--OPinac                                                                     19.0    2.0   2.34                                   43   Z-D-Phe--Pro--BoroOct--OPinac                                                                     22.5    3.86                                         51   Z-D-Dpa--Pro--BoroMpg--OPin                                                                       3.4     0.629 1.96                                   59   Z-D-Phe--Pro--BoroMbg--OPin                                                                       7.43    1.12  4.0                                    ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                                In-vitroassay  Km(μM)                                              ______________________________________                                                  H-D-Phe--Pip--Arg--pNA                                                                         10.0                                               34        H-D-β-Nal--Pip--Arg--pNA                                                                  6.9                                                35        H-D,L-Dpa--Pip--Arg--pNA                                                                       9.1                                                ______________________________________                                    

Those compounds of the invention which are thrombin inhibitors haveanti-thrombogenic properties and may be employed for indications when ananti-thrombogenic agent is indicated. Generally, these compounds may beadministered orally or parenterally to a host to obtain ananti-thrombogenic effect. In the case of larger mammals such as humans,the compounds may be administered alone or in combination withpharmaceutical carrier or diluent at a dose of from 0.02 to 15 mg/Kg ofbody weight and preferably 1-10 mg/Kg to obtain the anti-thrombogeniceffect, and may be given as single dose or in divided doses or as asustained for a patient, 0.1-1 mg/Kg may be administered intravenously.For use with whole blood from 1-10 mg per liter may be provided toprevent coagulation. Pharmaceutical diluents are well known and includesugars, starches and water which may be used to make tablets, capsules,injectable solutions and the like. The compounds of the invention may beadded to blood for the purpose of preventing coagulation of the blood inblood collecting or distribution containers, tubing or implantableapparatus which comes in contact with blood.

The advantages of the compounds of the invention include oral activity,rapid onset of activity and low toxicity. In addition, these compoundsmay have special utility in the treatment of individuals who arehypersensitive to compounds such as heparin.

In the following examples, the symbols have the following meanings:

Aa=amino acid

Ac=acetyl

Boc=t-butyloxycarbonyl

Bu=butyl

Bzl=benzyl

Bzl=Benzyl

DCC=dicyclohexylcarbodiimide

DIEA=diisopropylethylamine

DMAP=4-dimethylaminopyridine

EtOAc=ethyl acetate

EtOH=ethylalcohol

HOSu=N-hydroxy succinimide

MCA=4-methyl-coumaryl-7-amide

MeOH=methylalcohol

Mtr=4-methoxy-2,3,6-trimethylbenzenesulphonyl

NMR=nuclear magnetic resonance

NP=p-nitrophenyl

PinOH=pinanediol

PfpOH=pentafluorophenol

pip=piperidide

pNA=p-nitroanilide

TLC=thin layer chromatography

THF=tetrahydrofuran

TEA=triethylamine

WSC=water soluble carbodiimide

Z=Cbz=benzyloxycarbonyl

Apa=amidinophenylalanine

Chg=cyclohexylglycine

Dpa=3,3-diphenylalanine

Gpa=guanidinophenylalanine

Irg=isothiouronium analogue of Arg

ArgCN=Arg, where COOH is replaced by CN

Mbg=2-(2-methylbutyl) glycine

Nal=naphthylalanine

Pgl=pentylglycine

Thi=thiazolidinecarboxylic acid

boroAa=boronic acid analogue of Aa

Aa^(P) =phosphonic acid analogue of Aa

--k--=amide bond replaced by CO₋ Ch₂

The following non-limiting examples illustrate the preparation of thecompounds in this invention.

The synthesis of some of the different inhibitor types are outline inSchemes 1 to 8 and the detailed descriptions are given in the examplesbelow.

HPLC

The following conditions were adopted for the analysis of most of thesynthetic compounds on reversed-phase HPLC (RP-HPLC): column; SuperPacPep-S (4×250 mm), eluant; A=water containing 0.1% TFA, B=acetonitrilecontaining 0.1% TFA, gradient; 50% to 90% B in A in 25 min, flow rate;1.0 ml/min, detection; UV absorbance at 210 nm.

TLC

Thin layer chromatography (TLC) was carried out on the followingcompounds using precoated silica plates (Merck, F254) in the followingsystems: A, Chloroform-ethyl acetate (2:1); B,chloroform-methanol-acetic acid (20:4:1); C, n-butanol-acetic acid-ethylacetate-water (1:1:1:1); D, chloroform-methanol (9:1); E, pyridin-ethylacetate-acetic acid-water (5:5:1:3); F, chloroform-methanol-ammonia (1M)(60:35:5). The spots were visualized by ninhydrin andchlorine-dicarboxidine spray reagents (C. M. Swahn and J. Gyllander, J.Chromatogr. (1970) 170, 292:

NMR spectra

Magnetic resonance spectra were recorded at 250 MHz using a Brukerinstrument.

EXAMPLES 1. Synthesis of Dpa, Z-Boc-Dpa-Pro and Z-Dpa-Pro-Arg (Cf.Scheme 1)

(a) DL-Dpa.HCl

To a solution of potassium tertiary butoxide (6.75 g, 0.06 mol) intertiary butanol (350 ml) was added, at room temperature under argon,ethyl acetamido cyanoaetate (10 g, 0.059 mol). When the solution hadbecome clear bromodiphenyl methane (14.55 g, 0.059 mol) was added. Themixture was stirred at 20° C. for 24 h, then evaporated under reducedpressure. The solid residue was treated with ethyl acetate (500 ml) andwater (175 ml). The organic phase was dried (Na₂ SO₄) and concentratedto give yellow crystals. The crystals were washed repeatedly with etherand dried to give ethyl 2-diphenylmethylacetamido cyanoacetate (11.61 g,58%, m.p. 181°-185° C.). The ester (11.61 g, 34.4 mol) was mixed withhydrochloric acid (20%) and refluxed for 30 h. The reaction mixture wasallowed to cool and the crystals were collected, washed (ether), anddried to give HCl.D,L-Dpa (7.82 g, 81.8%).

(b) Z-DL-Dpa

To a solution of D,L-Dpa.HCl (0.56 g, 0.0021 mol) in NaOH (2N, 5 ml),cooled to 0° C. and vigorously stirred was added, dropwise, benzylchloroformate (0.39 g, 0.33 ml, 0.0023 mol). The reaction mixture waskept at pH10 and at 5° C. to 10° C. The solution was warmed to roomtemperature and stirred vigorously for 1 h. The solution was washed withether (4 times) and acidified to pH3 with HCl (5N). The mixture wasextracted with dichloromethane and the organic phase dried andconcentrated to give Z-DL-Dpa (0.73 g, 97%,, m.p. 214°-217° C.).

(c) Z-D,L-Dpa-ONSu

To a stirred solution of Z-D,L-Dpa (1.88 g, 0.005 mol) and N-hydroxysuccinimide (0.575 g, 0.005 mol) in dry 1,2-dimethoxyethane (30 ml) at0° C. was added dicyclohexyl carbodiimide (1.03 g, 0.005 mol). Themixture was maintained at 0° C. for 4 h. The suspension was filtered andthe filtrate was concentrated to dryness to give an oil which wastriturated with ether and filtered to give Z-D,L-Dpa-ONSu (2.15 g, 91%,m.p. 139°-142° C.).

(d) Z-D-Dpa-Pro and Z-L-Dpa-Pro

To a solution of proline (0.78 g, 0.0068 mol) and NaHCO₃ (0.57 g, 0.0068mol) in water (8 ml) was added a solution of Z-D,L-Dpa-ONSu (2.15 g,0.0045 mol) in 1,2-dimethoxyethane (15 ml). After 2 h the solvent wasremoved under reduced pressure and water (5 ml) was added. The solutionwas acidified (conc. HCl) to pH2 to give white crystals (1.98 g, m.p.113°-117° C.). Fractional recrystallisation from EtOAc gave as the firstcrop one diastereomer as a solid (0.7 g, m.p. 180°-183° C., FAB MS:M⁺473; ¹ H nmr: 7.26 (15H, m, 3×Ph), 5.66(1H, d, CH), 5.23 (1H, m, CH),4.40 (1H, d, CH), 2.03 (2H, s, CH₂), 2.20 (4H, m, 2×CH₂); ¹³ Cnmr:172.19 (CO), 156.1 (CO), 139.17 (CO), 127-128 (Ph), 66.88 (CH₂),59.48 (CH), 55.58 (CH), 24.15 (CH₂). Further crystallisation from themother liquor gave as a second crop a mixture of diastereomers (0.43 g,m.p. 126°-130° C.). Addition of petroleum ether (b.p. 60°-80° C.) gavethe other isomer (0.54 g, m.p. 128°-131° C.), FAB MS:M³⁰ 473; H nmr:7.29 (15H, m, 3Ph), 5.55 (1H, d, CH), 5.23 (1H, m, CH), 4.47 (1H, d,CH), 2.04 CH₂); ¹³ C:172.77 (CO), 156.13 (CO), 139.48 (CO),126.99-128.72 (Ph), 66.90 (CH₂), 59.62 (CH), 55.48 (CH), 53.54 (CH),47.44 (CH₂), 27.94 (CH₂), 24.58 (CH₂).

(e) Z-D-Dpa-Pro-Arg(Mtr)OPh

To a solution of Z-D-Dpa-Pro-OH (0.472 g, 1 mmol) and HOSu (0.115 g, 1mmol) in dimethoxyethane (20 ml) was added DCC (0.206 g, 1 mmol) withcooling over an ice water bath, the solution was then stirred at r.t.for 3 h, the DCU formed was filtered off and the solution wasconcentrated to dryness to give an oil (0.57 g). To a solution ofH-Arg(Mtr)-OH (0.42 g, 1.1 mmol) and Et₃ N (0.12 g, 1.1 mmol) in DMF (25ml) was added a solution of Z-Dpa-Pro-OSu (0.57 g) in dimethoxyethane(15 ml) with cooling. The solution was stirred at room temperature for 3h. The solvent was evaporated and the residue was dissolved in H₂ O (20ml) and MeOH (10 ml). The solution was acidified to pH2 and the MeOH wasremoved under reduced pressure. The solid formed was filtered off anddried to give Z-D-Dpa-Pro-Arg(Mtr)OH (0.766 g, 91%). The structure ofthe compound was confirmed by ¹ H NMR.

Fgl and Nal and their corresponding di- and tripeptides were synthesizedin a manner analogous to the above procedures.

2. Synthesis of peptide aminophosphonic acid inhibitors (Cf. Scheme 3)

(a) Diphenyl 1-(N-benzyloxycarbonyl)aminopentanephosphonate

A mixture of triphenyl phosphite (9.3 g, 30 mmol), n-hexanal (4.50 g, 45mmol), benzylcarbamate (4.53 g, 30 mmol), glacial acetic acid (5 ml) wasstirred for 45 min. The mixture was then heated at 80°-85° C. for 1 hand volatile by-products were removed in vacuo with heating on a boilingwater bath. The oily residue was dissolved in methanol (40 ml) and leftfor crystallization at -10° C. to give 7.28 g, m.p. 70°-72° C., 52%yield. The structure was confirmed by proton NMR.

(b) Diphenyl 1-aminopentanephosphonate

Diphenyl-1-(N-benzyloxycarbonyl) aminopentanephosphonate (0.93 g, 2.0mmol) was dissolved in ethanol (30 ml) and acetic acid (0.2 ml) wasadded. Then 10% palladium on charcoal (100 mg) was added and the mixturewas hydrogenated for 4 h. The catalyst was filtered off, washed withethanol (5×5 ml). After removal of the solvent an oil was obtained. Theoil was washed with water to remove acetic acid and dissolved inchloroform, dried (MgSO₄), concentrated to dryness to give oily product,0.45 g, 68% yield. The structure was confirmed by proton NMR and MS.

(c) Z-D-Dpa-Pro-Pgl^(P) (OPh)₂

Z-D-Dpa-Pro-OH (0.11 g, 0.25 mmol) was dissolved in dry chloroform (2ml) containing Et₃ N (0.035 ml) was cooled to -5° C. Ethyl chloroformate(0.026 ml, 0.275 mmol) was added and the mixture kept at -5° C. for 30min. A solution of diphenyl 1-aminopentanephosphonate (83 mg, 0.25 mmol)in dry chloroform (2 ml) containing Et₃ N (0.025 g, 0.25 mmol) wasadded. The mixture was stirred at r.t. for 12 h. Solvent was removed invacuo. The resulting oil was chromatographed (CHCl₃ then 2% MeOH inCHCl₃) to give 123 mg as crystals, 63% yield. The structure wasconfirmed by proton and ³¹ p NMR.

(d) H-D-Dpa-Pro-Pgl^(P) (OPh)₂

Z-D-Dpa-Pro-Pgl^(P) (OPh)₂ (50 mg, 0.063 mmol) was dissolved in ethanol(5 ml) and acetic acid (0.01 ml) was added. 10% Pd/C (25 mg) was addedand the mixture was hydrogenated at r.t. for 3 h. The catalyst wasfiltered off and ethanol removed in vacuo. The resulting oil was treatedwith water (5 ml) and chloroform (20 ml). The chloroform phase was dried(MgSO₄) and concentrated to dryness to give crystals, 41 mg, 91% yield.The structure was confirmed by ¹ H and ³¹ P NMR.

(e) H-D-Dpa-Pro-Pgl^(P) (CH)₂

Z-D-Dpa-Pro-Pgl^(P) (OPh)₂ (100 mg, 0.127 mmol) was dissolved in ethanol(10 ml) and acetic acid (0.1 ml) was added. Then 10% Pd/C (50 mg) wasadded and the mixture was hydrogenated at r.t. for 3 h. The catalyst wasfiltered off, PtO₂ (100 mg) was added and the mixture was hydrogenatedat r.t. for 4 h. The catalyst was filtered off, solvent was removed andthe residue was treated with 20 ml of water and chloroform (60 ml). Theorganic layer was dried (MgSO₄), concentrated to dryness to give 67 mgas crystals, 92% as overall yield. The structure was confirmed by ¹ Hand ³¹ P NMR.

Z-D-Phe-Pro-Pgl^(P) (OPh)₂

This compound was synthesized by the above procedure in 73% yield. Thestructure was confirmed by ¹ H and ³¹ P NMR.

H-D-Phe-Pro-Pgl^(P) (OPh)₂

The compound was synthesized by the above procedure in 90% yield. Thestructure was confirmed by ¹ H and ³¹ P NMR.

H-D-Phe-Pro-Pgl^(P) (OH)₂

The compound was synthesized by the above procedure in 89% overallyield. The structure was confirmed by ¹ H and ³¹ P NMR.

(f) Diphenyl 1-(N-allyl)amino-4-Pyridylmethyl-phosphonate

To a solution of 4-pyridinecarboxyaldehyde (1.07 g, 10 mmol) andallylamine (0.61 g, 10 mmol) in ether (30 ml) was added anhydrous sodiumcarbonate (2.76 g). The solution was stirred at r.t. overnight, thensodium carbonate was filtered off. To the reaction mixture,diphenylphosphite (2.34 g, 10 mmol) and triethylamine (1.01 g, 10 mmol)were added with cooling over an ice-water bath. It was stirred at r.t.overnight. After removal of the solvent an oily residue was obtainedwhich was chromatographed (1:1 Petroleum ether/ethyl acetate) to give2.85 g (65%) as an oil.

(g) Diphenyl1-(N-allyl)amino-4-(tert-butyloxycarbonyl)butyl-phospohonate

4-(tert-butyloxycarbonyl)amino-butylaldehyde diethyl acetal (2.91 g, 10mmol) was dissolved in acetone (20 ml) in the presence of 1N hydrogenchloride (1 ml) and PPTS (150 mg). The reaction mixture was refluxed for3 h. The solvent was removed and the residue was dissolved in chloroformand dried (MgSO₄). After removal of MgSO₄ the solution was stirred andallylamine (0.61 g, 10 mmol) and anhydrous sodium carbonate (2.76 g)were added. The reaction suspension was stirred at r.t. overnight. Thesodium carobonate was filtered off. To the solution obtained,diphenylphosphite (2.34 g, 10 mmol) and the triethylamine (1.01 g, 10mmol) were added. It was stirred at r.t. for 2 days. The residueobtained after evaporation was chromatographed on silica gel (1:1Petroleum/ethyl acetate) to give 200 mg as a yellow waxy solid (5%).

(h) Diphenyl 1-amino-4-pyridyl-methyl-phosphonate

The N-allyl protected compound (1.0 g, 2.3 mmol) was dissolved inethanol (25 ml). To the solution was added 10% Pd/C (300 mg) that it wasrefluxed for 20 hours. The reaction was followed by HPLC, retentiontime; 10.0 min for the product and 12.0 min for the starting material.After removal of the solvent the product was obtained by chromatography,0.51 g (56%) as a yellow oil.

The following tripeptides were synthesized according to Scheme 5:

Z-D-Phe-Pro-Cpg^(P) (OPh)₂ :0.36 g(67%)

Z-D-Phe-Pro-Epg^(P) (OPh)₂ :0.31 g(87%)

Z-D-Phe-Pro-Pyg^(P) (OPh)₂ :0.41 g(35%)

Z-D-Phe-Pro-Dmg^(P) (OPh)₂ :0.25 g(70%)

Z-D-β-Nal-Pro-Mpg^(P) (OPh)₂ :54 mg(32%)

H-D-Phe-Pro-Epg^(P) (OPh)₂ :126 mg(71%)

H-D-Phe-Pro-Dmg^(P) (OPh)₂ :85 mg(52%)

Abbreviations:

Mpg=methoxypropylglycine

Cpg=4-cyanophenylglycine

Epg=2-ethylpropylglycine

Pyg=4-pyridylglycine

Dmg=3,3-dimethylpropylglycine

Nal=naphtylalanine

PPTS=Pyridinium p-toluenesulfonate

3. Synthesis of peptide aminoboronic acid inhibitors

(a) (+)-Pinanediol 4-bromo-R-1-aminobutane boronate hydrochloride

The title compound was prepared as described by D. S. Matteson et al(1984) in Organometallics, 3, 1284-1288 and in European patent appl.293881A2.

(b) Z-D-Dpa-Pro-Irg-OPin.HCl

To a solution of Z-D-Dpa-Pro-OH (236 mg, 0.5 mmol) in THF (5 ml) in thepresence of trieethylamine (70 μl, 0.5 mmol) was added isobutylchloroformate (65 μl, 0.5 mmol) at -15° C. and the solution was stirredat -13° C. for 13 min. After the addition of (+)-pinanediol4-bromo-R-1-aminobutaneboronate hydrochloride (183 mg, 0.5 mmol) inCHCl₃ followed by that of Et₃ N (70 μl, 0.5 mmol), the reaction mixturewas stirred at the same temperature for 2 h, and then below 10° C. for 2h. THF was removed under reduced pressure and the residue was dissolvedin ethyl acetate (50 ml), which was washed with 1% NaHCO₃, water, 0.2NHCl and water, and then dried over Na₂ SO₄. Removal of solvent gave anoily product quantitatively. HPLC analysis showed one major peak at theretention time of 22.8 min along with several minor components.

To a solution of the above compound (2/5th of total amount synthesized,0.2 mmol) in ethanol (1 ml) was added chiourea (61 mg, 0.8 mmol) underan atmosphere of argon at room temperature. After stirring for 4 days,ethyl acetate (70 ml) was added to the reaction mixture, which waswashed with 1% NaHCO₃, water, 0.2N HCl and then water, and dried overNa₂ SO₄. The residue obtained by removing the solvent was treated withn-hexane to get the product as a powder. Reprecipitation from ethylacetate with 2:1 mixture of ethyl ether and n-hexane gave a product(98.8 mg, 60.6%, two step overall). Retention time on RP-HPLC analysiswas 13.5 min under the conditions described at the general procedure. ¹H NMR analysis is deuterated chloroform gave a complex pattern becauseof the existence of proline of proline residue in the molecule, however,the typical signals corresponding to pinanedioil were observed as properratios.

4. Z-D-Phe-Pro-boroMbg-OPin (Cf. Scheme 4)

A solution of pinanediol (dichloromethyl)boronate (1 ml, 1.2 g, 4.6mmol) in THF (7 ml) was placed in a septum fitted flask (100 ml), and1,1-dimethylpropane magnesium chloride (4.6 ml, 4.6 mmol) added dropwisefrom a dry syringe at 0° C.

The reaction mixture was left stirring under nitrogen at roomtemperature. After 7 hours TLC showed mainly one spot [Rf=0.82,chloroform:pet.ether (1:1)]. The solvent was removed and the residuedissolved in ether (50 ml), washed with water (2×10 ml), dried (MgSO₄)and filtered.

The ether was removed and the crude product purified on a column ofsilica gel, eluted with hexane and 10% of chloroform to giveα-chloroboronic ester as a pale yellow oil (0.55 g, 40% yield).

The above compound (0.55 g, 1.8 mmol) in THF (5 ml) was added via adouble ended needle at -78° C. to a solution oflithiumbis(trimethyl-silyl) amide (1.8 ml, 1.8 mmol) in THF (5 ml) undernitrogen. The reaction mixture was kept overnight at 20° C. then thesolvent was removed. The crude product was dissolved in petroleum ether(40°-60° C.) (25 ml) to precipiate out the inorganic salt (LiCl). Thereaction mixture was filtered, cooled to -78° C. and dry etheral HCl, 1M(3 equiv, 5.4 mmol) added. The flask was kept in a fridge overnight.Next morning, the reaction mixture was filtered to isolate thehydrochloride (0.41 g, 1.29 mmol, 72% yield) as a white solid.

Z-D-Phe-Pro-OH (0.45 g, 1.1 mmol) was dissolved in THF (7 ml) and theequivalent of N-methylmorpholine (0.11 g, 1.1 mmol) added. The solutionwas cooled to -20° C. and one equivalent of isobutylchloroformate (0.148g. 1.1 mmol) added dropwise. After 10 min., a solution of the aboveaminohydrochloride (0.348 g, 1.1 mmol) dissolved in THF (7 ml) wastransferred under nitrogen, and triethylamine (0.11 g, 1.1 mmol) addedto the reaction mixture. The reaction mixture was stirred for one hourat -20° C., followed by 2 h at room temperature. Insoluble material wasremoved by filtration, then the solvent removed by evaporation, and theresidue dissolved in ethyl acetate (30 ml). The organic layer was washedwith 0.2N hydrochloric acid (10 ml), 5% aqueous sodium bicarbonate,saturated solution of sodium chloride and water. The organic phase wasthen dried over anhydrous MgSO₄, filtered and the solvent evaporated togive a white solid which was purified on a column of silica get elutedwith light petroleum to give the desired product (0.59 g, 81%). Thestructure was confirmed by ¹ H NMR and MS.

5. PREPARATION OF α-BROMO BORONIC ESTERS (Cf. Scheme 6)

All the reactions involving boron used purified anhydrous reagents.

Reactions were carried out under argon or nitrogen used directly fromthe cylinder through a glass line.

In a 250 ml reaction flask fitted with a reflux condenser was placed1-bromo-1-propene (3.63 g, 30 mmol).

Dibromo borane -methyl sulfide complex in dichloromethane (60 ml, 60mmol) was then added to the reaction flask dropwise and the mixture wasrefluxed under nitrogen for 5 h.

The solvent was removed and the reaction mixture washed with water anddried (MgSO₄).

A dry round-bottomed flask (100 ml) was charged with the bromo boronicacid (0.5 g, 3 mmol) and pinanediol (0.52 g, 3 mmol) a magnetic followerand dry ether (20 ml), fitted with a septum and flashed with nitrogen.

The reaction mixture was left stirring for two hours until the soliddissolved, the organic phase was washed with water (10 ml), separated,dried (MgSO₄) and filtered. The crude product was purified on a columnof silica gel (230-400 mesh), eluted with chloroform (the product waseluted before the pale red ring). The first fraction (100 ml) wascollected and the solvent evaporated to give α-bromo boronic ester (0.8g, 88.6%) as a colourless liquid.

6. Synthesis of isosteric ketomethylene inhibitors (Cf. Scheme 2)

(a) Boc-D-Dpa-Pro-Arg(Mtr)-k-GlyOMe (modified Darkin-West reaction)

Boc-D-Dpa-Pro-Arg(Mtr)-OH (0.126 g, 0.15 mmol) was added tomonomethylsuccinyl anhydride (0.259 g, 1.0 mmol). Et₃ N (0.042 ml, 0.30mmol), DMAP (1.8 mg, 0.015 mmol) and pyridine (0.12 ml) were added, thereaction flask was fitted with a reflux condenser, and the reaction washeated at 45°-50° C. The reaction mixture was stirred for 1 h, thenNaHCO₃ (5%, 5 ml) was added and the stirring was continued for anadditional 30 min. The product was extracted into ethyl acetate andwashed with AcOH (0.1N) and brine. The organic layer was dried (MgSO₄),filtered and concentrated to dryness to give an oily residue which waschromatographed on silica gel (grade 9385, 50 g). Elution with CHCl₂:CH₃ OH,98:2 gave the product after removal of the solvent as a brownoil (0.134 g, 98%). The structure was confirmed asBoc-Dpa-Pro-Arg(Mtr)-k-GlyOMe by ¹ H NMR (250 MHz), and by FAB massspectrometry.

(b) Z-D-Dpa-Pro-Arg(Mtr)-k-Gly-pip

A solution of Z-D-Dpa-Pro-Arg(Mtr)-k-Gly-OMe (0.1 g, 0.1 mmol) in MeOH(10 ml) was cooled to 0° C. and NaOH (1N, 0.22 ml, 0.22 mmol) was addedwith stirring for 2.5 h at room temperature. The solution wasneutralized to pH7 and the MeOH was removed under reduced pressure. Theaqueous solution was acidified (pH2) and extracted by ethyl acetate anddried (Na₂ SO₄). The solvent was removed under reduced pressure to givean oil. To a solution of the oil and HOSu (12 mg, 0.1 mmol) in dimethoxyethane (20 ml) was added DCC (21 mg, 0.1 mmol), with cooling. Thesolution was stirred at room temperature for 20 h, and piperidine (17mg, 0.2 mmol) was added to the solution with cooling and the solutionwas stirred at room temperature for a further 3 h. The solution wasconcentrated to dryness and the product was purified by chromatographyon silica gel (MeOH:CHCl₃, 92:2) to give Z-D-Dpa-Pro-Arg(Mtr)-k-Gly-pip(78 mg, 81%). The structure of the product was confirmed by ¹ H NMR andFAB mass spectrometry.

In a separate experiment the L-isomer Z-L-Dpa-Pro-Arg(Mtr)-k-Gly-pip,was synthesized by the above procedure in 55% yield.

(c) H-D-Dpa-Pro-Arg-k-Gly-pip.2TFA

Z-D-Dpa-Pro-ArgMtr-k-Gly-pip (52 mg, 0.053 mmol) was dissolved in 0.9 mlof TFA and 0.1 ml of thioanisole at room temperature. After stirring for4 h. TFA was removed at reduced pressure and the residue was trituratedwith ether. The crystals were collected and washed with ether (52 mg ofMtr deprotected product). The crystals were then dissolved in 5 ml ofmethanol and 21 mg of 10% Pd/C was added. After 20 h. hydrogenation atroom temperature the catalyst was removed by filtration and the solventevaporated. The residue was triturated with ether to give whitecrystals. 34 mg (75%), m.p. 146-151 (dec.). HPLC showed two equally bigpeaks, from the two forms containing D and L Arg. The structure wasconfirmed by ¹ H NMR and FAB mass spectrometry.

In a separate experiment the L-isomer of Z-Dpa-Pro-Arg(Mtr)-k-Gly-pipwas deprotected to give H-L-Dpa-Pro-Arg-k-Gly-pip.2TFA in 43% yield.

7. Synthesis of peptide aldehydes (Cf. Scheme 8)

(a) Z(NO₂)Arg NCH₃ (OCH₃)

N,O-dimethylhydroxylamine hydrochloride (1.45 g, 14.9 mmol) wasdissolved in 10 ml of DMF, and the solution kept at 0° C.Diisopropylethylamine (1.92 g, 14.9 mmol) was first added and thenZ-(NO₂)Arg in 10 ml of DMF, HOBT (1.92 g, 14.2 mmol) and WSC.HCL (2.99g, 15.6 mmol). After 5 h reaction 0° C. the solution was left at roomtemperature overnight. The solvent was removed at reduced pressure and100 ml of EtOAc and 25 ml of H₂ O were added. The organic phase wasdiluted with ether and washed with Na₂ CO₃ (0.5M), H₂ O, H₂ SO₄ (0.1M)and H₂ O, then dried and the solvents removed giving 3.66 g of product.The water solutions were combined, extracted and treated in the same wayas above giving further 1.69 g. Totally 5.35 g (95%) which waschromatographed on Sephadex LH-20 with 95% ethanol. Yield 4.60 g (82%)of homogenous product (TLC in S₁ and S₂). NMR confirmed the structure.

(b) Arg(NO₂)--NCH₃ (OCH₃).HBr

The above compound was deprotected in HBr/HOAc at room temperature for45 min in the usual manner. The product was homogeous according to TLCin S₃, S₄ and S₅.

(c) Boc-D,L-Dpa-Pro-Arg(NO₂)--NCH₃ (OCH₃)

Arg(NO₂)--NCH₃ (OCH₃).HBr (4.9 g, 13 mmol) was dissolved in DMF, thesolution cooled to -5° C. and Et₃ N added to alkaline reaction.Boc-DL-Dpa-Pro OH (5.5 g, 12.5 mmol), HOBT (1.7 g, 12.5 mmol) and WSC(2.8 g, 14.5 mmol) were added and the reaction mixture stirred for 2 h,at -5° C. and then stirred at room temperature overnight. The solventwas evaporated at reduced pressure, EtOAc and H₂ O were added, theorganic phase separated and extracted with 0.5M NaHCO₃ (3×30 ml), NaClsolution (4×20 ml), dried (NaSO₄) and the solvent removed. Yield 8.3 g(97%) TLC (S₂) shows one spot. The expected structure of the compoundwas confirmed with NMR.

(d) Boc-D,L-Dpa-Pro-Arg-NCH₃ (OCH₃).HCl

The above compound (2.33 g, 3.4 mmol) was dissolved in 240 ml of MeOHand 3.6 ml HCl (1M). The catalyst, 10% Pd/C (0.6 g) was added andhydrogenation performed at room temperature for 20 h. The catalyst wasfiltered and the solvent removed at reduced pressure. Remaining 2.3 gsolid contained some starting material which was removed by ion exchangechromatography on Sephadex QAE⁺ Cl⁻ with 50% EtOH. Yield 1.74 g (76%).TLC (S₂ and S₃) showed a single spot.

(e) Boc-D,L-Dpa-Pro-Arg-H.HCl

The above compound (0.5 g, 0.74 mmol) was dissolved in 40 mol of dried(molecular sieve, 4A) THF and the solution was cooled to -40° C. and 3.2ml of DIBAH (1M toluene solution, 3.2 mmol) was added dropwise duringstirring in argon atmosphere. After 3 h, 12.8 ml of 0.25M citric acidwas added. The aluminium salts were centrifuged and washed several timeswith THF/H₂ O (4:1). From the combined liquid phases THF was removed atreduced pressure and the product extracted with EtOAc. The solvent wasremoved, the product dissolved in 15 ml of 20% HOAc and chromatographedon Sephadex G15 with 20% HOAc as eluent. Yield 172 mg (38%). Thecompound shows a double spot in TLC (S₂ and S₃) probably showing the twoisomeres with D- and L-Dpa, respectively. NMR and MS were in agreementwith the expected structure.

8. Synthesis of Boc-D,L-Dpa-Pro-ArgCN.HCl

(a) Z-D,L-Dpa-Pro-Arg-NH₂.HCL

Arg-NH₂.2HCl and Boc-D-L-Dpa-ProOH were coupled in the normal way withHOBT and DCC in DMF. Yield 53%.

(b) Boc-D,L-Dpa-Pro-ArgCN.HCl

The above tripeptide amide (0.50 g, 0.79 mmol) and tosylchloride (0.50g, 2.55 mmol) were dissolved in 2 ml of pyridine at room temperature andstirred for 24 h. The pyridine was evaporated at reduced pressure, 5 mlof pyridine and 0.5 ml of water were then added and stirring continuedfor 2 h. After evaporation at reduced pressure the residue wastriturated with a small amount of water, dissolved at EtOAc, dried (Na₂SO₄) and chromatographed on Sephadex QAE⁺ Cl⁻. The fractions containingthe product were evaporated, dissolved in 10% HOAc and freeze dried.Yield:0.33 g (68%). [α]D²²° =-144° (C=0.5, 50% HOAc). The structure wasconfirmed with ¹ H NMR and mass spectroscopy.

9. Synthesis of Dba

(a) Preparation of N-formyl-N,α,β-tribenzylalanine cyclohexylamide

Bzl-NH₂ 288 μl (2.64 mmol) and benzyl phenethylketone 592 mg (2.64 mmol)were dissolved in MeOH (5 ml) at rt and the solution was stirredovernight. To the mixture were added formic acid 99.6 μl (2.64 mmol) andcyclohexylisocyanide 298 μl (2.4 mmol) at rt, which was allowed to reactat rt for 2 weeks. Insoluble material in MeOH was collected byfiltration and washed with MeOH, ether and then n-hexane. The crudeproduct was recrystallized from CHCl₃ with the 2 to 1 mixture of etherand n-hexane. Yield 540 mg (48.2%), NMR in CDCl₃ ; δ=0.8-1.8 cyclohexyl(11H), δ=2.1-4.75 CH₂ (8H), δ=5.45-5.55 NH (1H), δ=7.0-7.4 phenyl (15H),δ=8.25 (main) and 8.4 (minor) formyl (1H).

(b) Preparation of Bzl-Dba

The fully protected Dba 400 mg (0.85 mmol) was dissolved in 2.5 ml ofTFA and 3 ml of 11N HCl and the solution was kept at 145° C. withwater-cooled condenser and stirred for 20 h. After removal of TFA, thepH of the solution was adjusted around 7 by addition of 10N NaOH.Further addition of ether gave a powder which was collected and washedwith ether. Yield 124 mg (40.4%).

10. Synthesis of Z-D-Phe-Pro-Pgl-H

(a) Pgl

Pentylglycine was obtained by the Strecker* synthesis, using hexanal, ina yield of 31.6%.

(b) Z-Pgl

To a solution of pentylglycine (0.5 g, 3.5 mmol) in a mixture of water(4 ml) and THF (4 ml) to give a 0.5M solution, in the presence oftriethylamine (0.64 ml, 1.22 eq.) was added Z-OSu (0.963 g, 3.86 mmol.)at room temperature, solution became clear after 15 min. TLC after 2 hindicated some starting material and so a further portion oftriethylamine (0.2 ml) and Z-OSu (200 mg) was added. TLC after a further2 h indicated no starting material and so the solution was poured ontowater (50 ml) and was extracted by CHCl₃ (50 ml). The organic phase waswashed by HCl (1M, 20 ml) and dried (MgSO₄) and concentrated to giveZ-Pgl (1.18 g) as a gummy solid, which was recrystallised (DCM/Petroleumether bp60°-80° C.) to a white crystalline solid (0.8 g). Structure wasconfirmed by ¹ H Nmr.

(c) Z-Pgl-NMe(OMe)

To a solution of hydroxylamine (184 mg, 1.05 eq.) was added DIPEA (0.33ml, 1.05 eq.), and after 5 min a solution of Z-Pgl (0.5 g, 1.8 mmol) theHOBT (0.242 mg, 1 eq.). The solution was cooled to -15° C. and asolution of WSCI.HCl (0.378 mg, 1.1 eq.) was added. The solution wasmaintained at -15° C. for 30 min. then allowed to warm to roomtemperature and pH adjusted to ˜pH4 by addition of acetic acid. TLCafter 16 h shows little starting material and so the solution was pouredonto NaHCO₃ (100 ml) and extracted by Et₂ O (50 ml). The Et₂ O phase waswashed by NaCl (20 ml). The aqueous phase was washed by Et₂ O, theorganic phases were combined and then concentrated to give Z-PglNMe(OMe)(324 mg). Structure confirmed by ¹ H Nmr and mass spec.

(d) PglNMe(OMe)

To a solution of Z-PglNMe(OMe) (324 mg) in MeOH (10 ml) was applied avacuum, then argon. Purging was repeated twice, but on the third timePd/C (˜0.5 g) was added. Purging was repeated twice more and on thethird time the vacuum was quenched by bubbling Hydrogen through thesolution. AcOH (0.5 ml) was the added. After 90 min TLC indicated nostarting material and so the solution was filtered (celite), washingwith a large volume of MeOH, and concentrated to give PglNMe(OMe)(380mg), as a gum. Structure was confirmed by ¹ H Nmr.

(e) Z-D-PheProPglNMe(OMe)

To a solution of Z-D-PhePro (0.187 mg, 1 eq.) in DMF (2 ml) was addedDIPEA (0.084 ml, 1 eq.) and PyBOP (0.25 mg, 1 eq.), with stirring underargon. After 10 min a solution of PglNMe(OMe) (0.09 mg, 0.479 mmol), inDMF (1 ml), was added. After 90 min TLC indicated no starting materialand so the solution was poured onto HCl (1N, 50 ml) and extracted by Et₂O (50 ml). The Et₂ O layer was washed by NaHCO₃ (50 ml, 1.2N) and NaCl(sat'd, 20 ml) and dried (MgSO₄). Repeated chromatography on silica gel(Merck 9385), eluting with CHCl₃ /MeOH gave Z-D-Phe-Pro-PglNMe(OMe) (200mg). Fab Ms shows 567 (10%, M+H), and 589 (100%, M+Na) as required, thestructure was also confirmed by ¹ H Nmr.

(f) Z-D-Phe-Pro-Pgl-H

To a solution of Z-D-PheProPglNMe(OMe) (33 mg) in THF (2 ml) at -40° C.was added Di-isobutylaluminium hydride (1N, 0.155 ml, 2.5 eq.), underargon. The solution was allowed to warm to room temperature and stirredfor 18 h. TLC showed no starting material and so was quenched by H₂ SO₄(1N, 0.5 ml) and stirred for 10 min. The aqueous phase was thenextracted by EtOAc (20 ml). the organic phase was dried (MgSO₄) andconcentrated to give Z-D-PheProPgl-H (31 mg). The structure wasconfirmed by ¹ H Nmr and mass spectrum, and the compound was sent forbiological testing, as compound number 33.

11. Z-L-Val-pNa

4-nitroaniline (67.5 g, 0.48 mol) was dissolved in pyridine (dried over4A sieves, 750 ml), and the solution was cooled down with ice. PCl₃(34.3 g, 0.25 mol) was dissolved in pyridine (350 ml) and added dropwiseto the nitroaniline solution. The solution was allowed to stand for 30min. at room temperature. To the solution was added a solution ofZZVal-OH (112 g, 0.44 mol) in pyrridine (250 ml). The reaction mixturewas stirred at room temperature for 1 week, then the pyridine wasremoved on a rotavapor. The residue was treated with NaHCO₃ (2%). Theproduct crystallized was dissolved in boiling EtOH (2000 ml, 95%) andthe hot solution was filtered and left to stand overnight at roomtemperature. The crystals were filtered giving Z-L-Val-pNA (116.9 g,71.6%).

12. (a) Boc-Dpa

Boc-Dpa was obtained in an analogous manner to the procedure of Examples1(a) and (b).

(b) Boc-Dpa-Pip-Arg-pNA (Cf. Scheme 7)

To a solution of Boc-Dpa.HCl (150 mg, 0.396 mmol) in DMF(5 ml) was addedTBTU (133.5 mg, 0.416 mmol, 1.05 eq.) and DIPEA (0.069 ml, 0.396 mmol, 1eq.), to give a solution of basic pH. PipArgpNA.TFA (238 mg, 0.396 mmol,1 eq.), was then added. pH of the solution was acidic so a furtherportion of DIPEA (0.05 ml) was added. After stirring for 16 h, TLCshowed a little starting material and so a further portion of DIPEA wasadded (0.034 ml, 0.5 eq.). TLC after a further 3 h showed no startingmaterial and so the solution was poured onto EtOAc (50 ml) and washedHCl(0.5N, 200 ml), dried (MgSO₄) and concentrated to give 430 mg of gum.Trituration with Et₂ and petroleum ether (b.p. 60°-°80° C.), over 5minutes, gave a powder which was filtered, avoiding drying in the air,to give a BocDpaPipArgpNA.TFA as a powder, 207 mg, 62% yield, Rf 20 minon Rp HPLC (pep-S, 35-70% MeCN+0.1%TFA, 25 min, 1 ml/min.).

(c) Dpa-Pip-Arg-pNA

To solid BocDpaPipArgpNA (100 mg, 0.119 mmol), was added TFA (2 ml),with cooling in an ice bath for 10 min. The ice bath was then removedand the solution stirred for a further 10 min. TLC indicated no startingmaterial, so the solution was concentrated under vacuum (oil pump).Washing with Et₂ O(150 ml), until the filtrate was neutral pH, gave apowder dried under vacuum to DpaPipArgpNA.2TFA, 92 mg, (90%), as apowder, of retention time (15.5 min on Rp HPLC pep-s, 4×250 mm, 35-70%MeCN +0.1% TFA).

(d) Boc-D-Nal-Pip-Arg-pNA

To a solution of Boc- -D-Nal(150 mg, 0.474 mmol) in DMF (5 ml) was addedTBTU (164 mg, 0.51 mmol, 1.05 eq.) and DIPEA (0.083 ml, 0.474 mol), 1eq.). PipArgpNA.TFA (295 mg, 0.474 mmol, 1 eq.) was then added. After 30min TLC, using the Sakaguchi reagent (8-hydroxyquinoline, Br₂, NaOH),indicated mainly starting material, and so DIPEA (0.083 ml, 1 eq.) wasadded. After 30 min. TLC indicated mainly starting material and so thereaction was diluted by EtOAc (50 ml) and washed by HCl (0.5N, 100 ml).The organic phase was dried (MgSO₄), and concentrated to give an oil(490 mg). Recrystallization from CHCl₃ /petroleum ether bp 60°-80°C./Et₂ O gave 90 mg, 20.1% yield, of Boc-Dpa-PipArgpNA.TFA as a powder.

(e) H-D-NalPipArgpNA.2TFA

To solid Boc- -D-NalPipArgpNa.TFA (50 mg, 0.055 mmol), cooled in an icebath, was added TFA (2 ml), with stirring. After 10 min the ice bath wasremoved, TLC after 30 min still showed some starting material and sosolution was stirred for a further 10 min, then concentrated (oil pump).Trituration with Et₂ O have a powder. The powder was washed with Et₂ O,until the eluant was neutral, dried overnight at room temperature togive H-D-NalPipArgpNA as a powder, 42 mg, 92% yield. Retention time 11.5min on Rp HPLC.

EXAMPLE 13

The following compounds were prepared by the route substantially asoutlined in Examples 2a to 2e:

    __________________________________________________________________________                       Physical data to                                                           Yield                                                                            confirm structure                                          __________________________________________________________________________    15                                                                              Boc-D,L-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                      96%                                                                              Nmr, Ms.                                                   16                                                                              D,L-Dpa--Pro--Pgl.sup.P (OH).sub.2                                          17                                                                              Z-D-Dpa--Pro--Pgl.sup.P (OPh).sub.2                                         18                                                                               D-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                           92%                                                                              Nmr, FABMs[M + H]502, m.p. 130-133.                        19                                                                              Z-D-Phe--Pro--Pgl.sup.P (OPh).sub.2                                         20                                                                              D-Phe--Pro--Pgl.sup.P (OPh).sub.2                                                           35%                                                                              Nmr, FABMs.                                                21                                                                              D-Phe--Pro--Pgl.sup.P (OH).sub.2                                            23                                                                              D-Dpa--Pro--Pgl.sup.P (OPh).sub.2                                           32                                                                              H-D-Dpa--Pro--Mpg.sup.P (OPh).sub.2                                                            H, .sup.31 PNmr, HPLC.                                     40                                                                              H-D-Phe--Pro--Mpg.sup.P (OPh).sub.2                                                            HPLC.                                                      55                                                                              H-D-Phe--Pro--Apg.sup.P (OPh).sub.2                                                            HPLC.                                                      56                                                                              H-D-Phe--Pro--Epg.sup.P (OPh).sub.2                                                         71%                                                           57                                                                              H-D-Phe--Pro--DPg.sup.P (Oph).sub.2                                                         52%                                                                              Nmr.                                                       __________________________________________________________________________

EXAMPLE 14

The following compounds were synthesised according substantially toExamples 2a to 2d:

    ______________________________________                                        Z-D-Phe--Pro--Epg.sup.P (OPh).sub.2                                                                87%                                                      Z-D-Phe--Pro--Cpg.sup.P (OPh).sub.2                                                                67%                                                      Z-D--Phe-Pro--Pyg.sup.P (OPh).sub.2                                                                35%                                                      Z-D-β--Nal--Pro--Mpg.sup.P (OPh).sub.2                                                        32%                                                      ______________________________________                                    

EXAMPLE 15

The following compound was synthesised according to Examples 2a and 2b:

Mty^(P) (OPh)₂

EXAMPLE 16

The following compounds were synthesised according substantially toExample 2f:

D-Phe-Pro-Pyg^(P) (OPh)₂

D-Phe-Pro-Aeg^(P) (Boc)(OPh)₂

D-Phe-Pro-Npg^(P) (OPh)₂

EXAMPLE 17

The following compounds were synthesised according substantially toExamples 6a to 6c:

    __________________________________________________________________________    4  D,L-Dpa--Pro--Arg----k----Gly--Pip                                         5  D-Phe--Pro--Gpa----k----Gly--Pip                                                                 81.21                                                                             Nmr, FABMs, m.p. 110-114.                           6  D-Dpa--Pro--Arg----k----Gly--Pip                                                                 74.5%                                                                             Nmr, FABMs, m.p. 146-151.                           7  L-Dpa--Pro--Arg----k----Gly--Pip                                                                 42.7%                                                                             Nmr, FABMs, m.p. 136-140.                           8  D-Fgl--Pro--Arg----k----Gly--Pip                                                                 81.3%                                                                             Nmr, FABMs.                                         9  D,L-α--Nal--Pro--Arg----k----Gly--Pip                                                      81% Nmr, FABMs, m.p. 123-127.                           14 D,L-β--Nal--Pro--Arg----k----Gly--Pip                                                       554 FABMS.                                              54 D--β--Nal--Pro--Arg----k----Gly--Pip                                                        414 FABMS.                                              __________________________________________________________________________

EXAMPLE 18

The following compounds were synthesised according substantially toExample 10:

    __________________________________________________________________________    33                                                                              Z-D-Phe--Pro--Pgl--H                                                                            99.0% Nmr, Fabms[M + H]508, 7%                            48                                                                              Z-D-Dpa--Pro--Pgl--H                                                                            quantitative                                                                        Nmr, FabMs[M + H]584, 35%                           53                                                                              Boc-D-Phe--Pro--His--H                                                                          90%   Nmr, FabMs[M + H]48%                                  Z-N--Me--Phe--Pro--Pgl--NMe(OMe)                                            __________________________________________________________________________

EXAMPLE 19

The following compounds were synthesised according substantially toExample 11:

    ______________________________________                                        34     H-D-β--Nal--Pip--Arg--pNA                                                                   90.6%   Nmr                                         35     H-D,L-Dpa--Pip--Arg--pNA                                                                         81%     Nmr                                                H-D-Phe--Pro--Phe--pNA                                                 ______________________________________                                    

EXAMPLE 20

The following compounds were synthesised according substantially toExample 4:

    ______________________________________                                        22     Z-D-Phe--Pro--BoroMbg--OPin                                                                       89%     Nmr                                        41     Z-D-Phe--Pro--BoroPhe--OPinac                                                                     68.6%   Nmr                                        59     Z-D-Phe--Pro--BoroMbg--OPin                                                                       90%     Nmr                                        ______________________________________                                    

EXAMPLE 21

The following compounds were synthesised according substantially toExample 3a:

    ______________________________________                                        10   Z-D-Phe--Pro--BoroAcet--OPinac                                                                     42%       Nmr                                       11   Z-D-Phe--Pro--BoroPgl--OPinac                                                                      41.5*     Nmr                                       39   Z-D-Dpa--Pro--BoroMpg--OPin                                              43   Z-D-Phe--Pro--BoroOct--OPinac                                                                      774       Nmr                                       51   Z-D-Dpa--Pro--BoroMpg--OPin                                                                        quantitative                                                                            Nmr                                       ______________________________________                                    

EXAMPLE 22

The following compounds were synthesised according substantially toExamples 3a and 3b:

    ______________________________________                                        12  Z-D-Dpa--Pro--BoroIrg--OPin                                                                       61%     Nmr, FABMs[M]780, 13%.                        26  Z-D-β--Nal--Pro--Borolrg--OPin                                                               41.8%   Nmr, FABMs[M + H]755, 10%.                    36  Z-D-Fgl--Pro--BoroIrg--OPin                                                                       49%     Nmr, FABMs[M + H]778, 11%.                    37  Ac-D-Dpa--Pro--BoroIrg--OPin                                                                       8.1%   Nmr, FABMs[M + H]689, 14%.                    38  Z-L-Dpa--Pro--Borolrg--OPin                                                                       39%     Nmr, FABMs[M + H]781, 12%.                    46  Z-D-Cha--Prb--BoroIrg--OPin                                                                       41%     Nmr, FABMs[M + H]711, 10%.                    ______________________________________                                         ##STR9##

Plasma thrombin time (TT)

A volume of 150 μl of citrated normal human plasma and 20 μl of bufferor sample were warmed at 37° C. for 1 min. Coagulation was started byadding 150 ul of freshly prepared bovine thrombin (5NIHu/ml saline) andthe coagulation time was recorded on a coagulometer.

A phosphate buffer, pH7.8, containing 0.1% bovine serum albumine and0.02% sodium azide was used. The samples were dissolved in DMSO anddiluted with the buffer. When no inhibitor was used DMSO was added tothe buffer to the same concentration as that used in the samples. Theinhibitor concentrations were plotted against the thrombin times in asemilogarithmic graph from which the inhibitor concentration that causeda doubling (40 sec) of the thrombin time was determined.

Determination of Ki

The inhibition of human α-thrombin was determined by the inhibition ofthe enzyme catalyzed hydrolysis of three different concentrations of thechromogenic substrate S-2238.

200 μl of sample or buffer and 50 μl of S-2238 were incubated at 37° C.for 1 min and 50 μl of human α-thrombin (0.25 NIHu/ml) was added. Theinitial rate of inhibited and uninhibited reactions were recorded at 405nm. The increase in optical density was plotted according to the methodof Lineweaver and Burke. The Km and apparent Km were determined and Kiwas calculated using the relationship: ##EQU1##

Cardiovascular effects

Cats weighing 2-3 kg were anaesthetized with Mebumal, given as anintraperitnoeal injection. The central blood pressure and heart ratewere recorded on a Grass Polygraph by means of a catheter inserted inthe femoral

DETERMINATION OF km

The km of substrates with human α-thrombin were determined by measuringthe absorbance at a series of dilutions of substrate. (page 753, LongmanScientific & Technical, 5th edition, 1989.)

DETERMINATION OF ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) FORIN-VITRO SAMPLES

A volume of 150 μl of citrated (3.2%) normal human plasma was incubatedat 37° C. with sample (20 μl) or buffer (20 μl, control) for 1 min. Tothe solution was added reconstituted "AUTOMATED APTT" (available fromOrganon Teknika, 0.1 ml). Each solution was activated at 37° C. for 5min.

After activation, calcium chloride (0.1 ml, 0.02M, prewarmed at 37° C.)was added and clot detection was timed using a "semiautomatedcoagulometer" (Nach, Schnicter und Gross).

IN-VIVO TOXICITY DATA

Deposition of ¹¹¹ indium-labelled platelets was monitored after 1 mg/kgintravenous doses, via a cannula in the marginal ear vein of New Zealandwhite rabbits, as outlined in G. R. May, C. M. Hero, K. D. BUTLER, C. P.PAGE, JOURNAL OF PHARMACOLOGICAL METHODS, 24, pp 1-35, 1990. Peripheralblood pressure was monitored by a pressure sensor in the carotid artery.

EX-VIVO APTT, TT

Data was obtained as for in-vitro tests, but using plasma samplesobtained at 0,1,10,30 and 60 minutes after dosing of 1 mg/kg, i.v. bolusinjection in a marginal ear vein in New Zealand white rabbits, whichwere anaesthetized from a canula in the carotid artery. ##STR10##

    __________________________________________________________________________    PLATELET ACCUMULATION                                                         __________________________________________________________________________                   (% OF NORMAL)           BLOOD PRESSURE                         Compound       LUNG       LEG          Δ% of NORMAL                     __________________________________________________________________________    4 mg/kg iv. DOSE                                                              No. 25 ZD-PheProBoroIrgOPin                                                                  FATAL PLATELET                                                                           FATAL ACCUMULATION                                                                         100%                                                  ACCUMULATION                                                                             100%                                                               100%                                                           No. 53 BocDPhePro His--H                                                                     NONE       NONE         0                                      No. 22 ZDPheProBoroMbgOPin                                                                   NONE       11%, TRANSIENT                                                                             0                                      No. 41 ZDPheProBoroPheOPin                                                                   NONE       NONE         21%                                    1 mg/kg i.v. DOSE                                                             No. 59 ZDPheProBoroMbgOPin                                                                   NONE       NONE         NONE                                   No. 25 ZDPheProBoroIrgOPin                                                                   44-40%, PROLONGED                                                                        44-40%       SMALL                                  No. 20 H-D-PheProPgl.sup.P (OPh).sub.2                                                       NONE       NONE         NONE                                   No. 53 BocDPheProHis--H                                                                      NONE       NONE         NONE                                   No. 6 H-D-DpaProArg-k-Gly-pip                                                                NONE       NONE         NONE                                   __________________________________________________________________________    EX-VIVO DATA                                                                             T.T.              A.P.T.T.                                         ANIMAL     1       2         1         2                                      __________________________________________________________________________    COMPOUND NO. 6.                                                               TIME (MIN)                                                                     0         24.4                                                                              25.6                                                                              32.9  2.7.9                                                                             24        57                                      1         176 200 260   >200                                                                              46        78                                     10         55  61.4                                                                              76    57.5                                                                              32        55                                     30         33  36.9                                                                              62    36  34        57                                     60         25  27.9                                                                              49    31.5                                                                              27        42                                     COMPOUND NO. 11                                                               TIME (MIN)                                                                     0         30  24.5                                                                              26        46.5      26.4                                    1         >300                                                                              >200                                                                              >300      UC        UC                                     10         >300                                                                              >200                                                                              >300      UC        UC                                     30         102 >200                                                                              >300      236.7     49                                     60         94  >200                                                                              >300      53.0      38.4                                   COMPOUND NO. 59                                                               TIME (MIN)                                                                     0         28.9                                                                              22.5                                                                              22.4  23.5                                                                              42  59.5  43.9                                                                              58                                  1         111 106 171.5 168.5                                                                             111.4                                                                             111   102.9                                                                             126                                10         35.9                                                                              34  37.9  38.4                                                                              48.9                                                                              53    60.5                                                                              67                                 30         29  30  32    31  34.9                                                                              49    59  53.9                               60         28.4                                                                              28.5                                                                              27.9  28  40.4                                                                              48.4  65.5                                                                              62                                 COMPOUND NO. 53                                                               TIME (MIN)                                                                     0         26  27  24    24.9                                                                              32.9      24.9                                    1         86.9                                                                              83.5                                                                              80.9  74  41.5                                                                              41.5  27.9                                                                              30.9                               10         32  32  30    29.9                                                                              39.9                                                                              29.9  26  25.9                               30         24.4                                                                              25  25.9  25.4                                                                              31.4                                                                              30.4  25.4                                                                              26.1                               60         24.4                                                                              23.9                                                                              22.9  22.9                                                                              28.9      27.5                                                                              25.9                               __________________________________________________________________________

    __________________________________________________________________________                      IC50       Plasmin Xa    Urokinase                                                                           Kallikrein                                                                           Chymotrypsin                            No incubation                                                                       Inc. no inc/inc                                                                            no inc/inc                                                                          no inc/inc                                                                          no inc/inc                                                                           no                    __________________________________________________________________________                                                            inc/inc               15 Boc-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                           5.28  0.0836                                                16 D,L-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                           5.16  0.0817                                                18 D-Dpa--Pro--Pgl.sup.P (OH).sub.2                                                             148.0 0.151                                                 19 Z-D-Phs--Pro--Pgl.sup.P (OPh).sub.2                                                          149.0 0.0187                                                21 D-Phe--Pro--Pgl.sup.P (OH).sub.2                                                             non(137.0)                                                                          4.35                                                  23 D-Dpa--Pro--Pgl.sup.P (OPh).sub.2                                                            0.187 0.000938                                              32 H-D-Dpa--Pro--Mpg.sup.P (OPh).sub.2                                                          1.48  0.00235                                                                            n(187.0)/3.32                                                                         39.7/n18.7                                                                          83.0/n18.7                                                                          66.7/n18.7                                                                           n18.7/0.000149        40 H-D-Phe--Pro--Mpg.sup.P (OPh).sub.2                                                          16.6  0.0118                                                                             n20.9/52.5                                                                            n20.9/n20.9                                                                         n20.9/n20.9                                                                         n20.9/n20.9                                                                          n20.9/0.00263         55 H-D-Phe--Pro--Apg.sup.P (OPh).sub.2                                                                3.96                                                  56 H-D-Phe--Pro--Epg.sup.P (OPh).sub.2                                                                0.117                                                 57 H-D-Phe--Pro--DPg.sup.P (Oph).sub.2                                                                0.0828                                                22 Z-D-Phe--Pro--BoroMbg--OPinac                                                                0.007      11.5    3.86  9.13  12.9                         41 Z-D-Phe--Pro--BoroPbg--OPinac                                                                0.0128     37.3    7.94  6.67  16.7                         50 Z-D-Phe--Pro--BoroMpg--OPin                                                                  0.0007     3.23    1.72  3.63  7.26                          6 D-Dpa--Pro--Arg--k--Gly--Pip                                                                 0.2        n(157.0)                                                                              149.0 494.0 199.0                        44 Bzl--Pro--BoroIrg--OPin                                                                      1.3        0.153   0.512 0.121 1.08                         25 Z-D-Phe--Pro--BoroIrg--OPin                                                                  1.5        0.0084  0.0141                                                                              0.075 0.334                        52 Boc-D-Phe--Pro--His--H    n(413.0)                                                                              n(413.0)                                                                            n(413.0)                                                                            non(413.0)                   __________________________________________________________________________

References

1. Claeson, G, and Aurell, L. Annals of New York Academy of Sciences,370, 79-811 (1981).

2. Bajusz, S., Barabas, E., Tolnay, P. et al. Int. J. Peptide ProteinRes. 12, 217-221 (1978).

3. Kettner, C. and Shaw, E. Thrombosis Research 14, 969-973 (1979).

4. Szelke, M. and Jones, D. M. U.S. Pat. No. 4,638,047-A.

5. Kettner, C. and Shenvi, A. B. European patent appl. 293881 (1988).

6. Stuber, W., Kosina, H. and Heimburger, N. Int. J. Peptide ProteinRes. 31, 63-70 (1988).

7. Kaiser, B,, Hauptmann, J. and Markwardt, F. Die Pharmacie 42, 119-121(1987).

We claim:
 1. A peptide of the formula ##STR11## wherein X=H, CH₃ or anN-protecting group;Y=C₃ -C₉ alkyl optionally substituted by C₁ -C₄alkoxy; ##STR12## where R₁ and R₂ taken together represent the residueof a diol; Aa₁ =Dpa, Nal or Dba; and Aa₂ =proline.
 2. A peptideaccording to claim 1 which is selected from thefollowing:Cbz-D-Dpa-Pro-boroPgl pinacol ester Ac-D-βNal-Pro-boroMbgpinanediol ester and Cbz-D-Dpa-Pro-boroMpg-pinanediol ester.
 3. Apeptide according to claim 1 which is a substrate of thrombin.
 4. Apeptide according to claim 1 which is an inhibitor of thrombin.
 5. Apeptide according to claim 1, wherein Aa1 is Dpa.
 6. A peptide accordingto claim 5, wherein Y-- is MeOCH₂ CH₂ CH₂ --.
 7. A peptide according toclaim 1, wherein R₁ and R₂ taken together represent the residue ofpinacol or pinanediol.
 8. A peptide according to claim 6, wherein R₁ andR₂ taken together represent the residue of pinacol or pinanediol.
 9. Apeptide according to claim 8, wherein R₁ and R₂ taken together representthe residue of pinacol.
 10. A peptide according to claim 9 wherein X isCbz.
 11. A pharmaceutical composition comprising a peptide according toclaim 1 and a pharmaceutically acceptable carrier or diluent.
 12. Apharmaceutical composition according to claim 11, wherein said peptidehas the formula: ##STR13## wherein X=H, CH₃ or an N-protecting group;##STR14## R₁ and R₂ taken together represent the residue of pinacol orpinanediol.
 13. A method of inhibiting thrombin in a mammalian host,comprising administering to the host a thrombin inhibiting effectiveamount of a peptide according to claim
 1. 14. A method according toclaim 13, wherein the peptide is administered in combination with apharmaceutically acceptable carrier or diluent.
 15. A method accordingto claim 14, wherein Aa1 is Dpa.
 16. A method according to claim 15,wherein Y-- .sbsp.MeOCH₂ CH₂ CH₂ --.
 17. A method according to claim 15,wherein R₁ and R₂ taken together represent the residue of pinacol orpinanediol.
 18. A method according to claim 16, wherein R₁ and R₂ takentogether represent the residue of pinacol or pinanediol.
 19. A methodaccording to claim 17, wherein R₁ and R₂ taken together represent theresidue of pinacol.
 20. A method according to claim 19 wherein X is Cbz.21. A method according to claim 13 wherein the peptide is selected fromthe following:Cbz-D-Dpa-Pro-boroPgl pinacol ester Ac-D-βNal-Pro-boroMbgpinanediol ester and Cbz-D-Dpa-Pro-boroMpg-pinanediol ester.
 22. Amethod according to claim 13, wherein the peptide is administered at adose of from 0.02 to 15 mg/kg of body weight.
 23. A method according toclaim 22, wherein the dose is from 1 to 10 mg/kg of body weight.
 24. Amethod according to claim 13, wherein the peptide is administered as asingle dose or in divided doses or as a sustained release formulation.25. A method according to claim 13, wherein the host is a human host.26. A method of preventing coagulation of mammalian blood, comprisingadding thereto a thrombin inhibiting effective amount of a peptideaccording to claim
 1. 27. A method according to claim 26, wherein theamount of peptide added is from 1 to 10 mg/liter.
 28. A method ofestablishing an extracorporeal blood loop for a patient, comprisingadministering intravenously from 0.1 to 1 mg/kg of body weight of apeptide according to claim
 1. 29. A method of inhibiting the activity ofthrombin comprising contacting thrombin with a peptide according toclaim 1.